How to map using the RI lines

The Recombinant Inbred (RI) lines were generated from a cross
between the Arabidopsis ecotypes Columbia and Landsberg erecta
(Lister and Dean, 1993) (with Columbia as the male parent). 300 lines were generated to be used for
mapping. A large number of markers have been mapped using
100 of these lines. Mapping a new marker using these 100 will
give an accurate map position relative to >1000 other markers
however a rough map position can be determined with a much
smaller number of these lines (20 - 30, see below). For very fine
mapping the additional 200 lines can be used but markers in the
area of interest would then need to be scored on the 200 lines.

One can only directly map something on these lines if it is
polymorphic between Columbia and Landsberg erecta
and its segregation is therefore scorable in the RI lines.
QuantitativeTrait Loci (QTL), ie. loci affecting quantitative traits,
can also be mapped as they are segregating in the RI population,
although these may not be obviously polymorphic in the parental
ecotypes.

Larger probes (ie. cosmid or lambda clones) clearly increase the
chances of finding an RFLP, however if many restriction
fragments hybridize to the probe scoring of the polymorphism
may be difficult, in addition the bands which are polymorphic
may be cross-hybridizing sequences that map to different
positions in the genome. Sequences flanking T-DNA/transposon
inserts or cDNAs (ie 0.1 - 3kb) may be too small to show RFLPs
at a reasonable frequency so many enzymes may need to be
tested.

Enzymes that have been most useful for finding RFLPs between
Columbia and Landsberg erecta are BclI, BglII, BstEII, CfoI,
DdeI, DraI, EcoRI, EcoRV, HindIII, SacI and XbaI. Other
restriction enzymes which could be tested are the six-base cutters
with AT-rich recognition sequences (as the Arabidopsis genome
is AT-rich). Alternatively restriction enzymes with four-base
recognition sequences can also be tested. Use of small fragments
to isolate larger fragments (from cosmid or lambda libraries)
should also help in finding an RFLP.

ii) Phenotypic differences

Two examples are:-

a) The erecta mutation.

Landsberg erecta carries the erecta
mutation and therefore has a short and erect stature, Columbia has
the wild-type gene and is therefore taller and less compact. This
phenotype is segregating in the RI population and was scored in
the RI lines, and the data compared to the segregation data of the
67 RFLP markers. This resulted in a map position of the
ERECTA gene relative to the RFLP markers.

b) Resistance/susceptibility to a fungal pathogen.

The downy mildew Peronospora parasitica infects Arabidopsis. For the
isolate No-Co, there is a differential resistance response, with
Columbia showing the sensitive phenotype (sporulation, no
hypersensitive response (HR)) and Landsberg erecta, showing
the resistant phenotype (HR/cell necrosis). This trait segregated as
a single locus with the Ler allele dominant for resistance. In order
to map the locus (RPP5) the RI lines were infected with the No-
Co isolate and the response, either resistant (L) or sensitive(C),
scored (see below). This data was compared to the segregation
data of the 67 RFLP markers enabling the map position of RPP5
to be determined (Parker et al, 1993).

iii) Biochemical differences

The length and structure of the side chains of several
glucosinolates vary between Columbia and Landsberg erecta.
These differences can be detected using gas chromatography.
Glucosinolate profiles of the RI lines were carried out and using
this data the map positions of genes affecting the glucosinolate
pathway were determined (Magrath et al 1994).

iv) Quantitative Trait Loci

A wide range of quantitative traits are segregating in the RI lines,
including stature, rosette size, leaf number and shape, and
flowering time. These traits were not necessarily different in the
parental ecotypes but result from the many new combinations of
alleles in the Columbia and Landsberg erecta genomes that have
been brought together in the RI lines. These measurements can be
used in conjunction with the segregation data for the RFLP
markers to position the multiple loci influencing these traits (C.
Lister and C. Dean, unpublished results; D. Marshall and M.
Kearsey, unpublished results).

NOTE: concerning markers g4715-a and g4715-b:

The g4715 cosmid probe
detects two mappable polymorphisms, only one of which was reported
earlier as "g4715" on chromosome I. However, the Dean/Lister data
includes both polymorphisms. "g4715-a" is identical to the original
"g4715" marker on I. "g4715-b", the newer marker, is on chromosome V.
The image file displaying the g4715 polymorphism shows "g4715-a".

Steps to Mapping a Locus

1. Identify an RFLP

Identify an RFLP or other polymorphic feature in the parental
ecotypes (see above) or a quantitative trait which can be scored in
the RI lines.

2. Sow out the RI lines.

Plants to be used for DNA preps can be
grown either in the glasshouse and tissue culture (for leaves) or in
liquid media in flasks (for mainly root material). Plants that are
grown for phenotypic or biochemical examination may require
special growing conditions or treatments to reveal/accentuate the
differences between Columbia and Landsberg erecta (ie
inoculation with fungus, different light regimes, etc..).

If only using 20-30 RI lines for mapping the
following lines have been selected as having the
highest frequency of recombination over the five
chromosomes and therefore should be the most
informative for mapping purposes:-

iii) Biochemical difference. Carry out the biochemical assay and
score.

(iv) QTL Count or measure the trait under examination, usually
recorded as a mean from 5-10 individuals/RI line.)

In all the above experiments do not forget to include the Columbia
and Landsberg erecta parents at the same time, as controls.

4. Submit the data to NASC.

Please note that this service is no longer available due to a rapid decrease in requests following the publication of the sequence. We hope this does not cause any problems and thanks to all of the users that populated this map in the past

The text below is only recorded for historical reasons

RI mapping is conducted using Mapmaker (Lander et al 1987) and so the data
has to conform to the Mapmaker format. We have designed an RI data submission form for data submission to help you send in the data in the correct format. We recommend that you use this form for data submission and that you fill in the information for each line. It has been designed to allow us to automatically enter your data and so circumvents any potential problems with typing errors.

However, we can also receive data by email
Label your message RIDATA. If sending your information by email please adhere
strictly to the following format, indicating the numbers of the RI lines
used and including the data in (RI) numerical order.

Note: concerning plant order:

The 101 plants in the Dean/Lister
population are presented in the following order. Each symbol
corresponds to one an RI line (for example, "4" corresponds to
CL4, which is Nottingham strain N1900.

PLEASE look at the RI data submission pages to correlate these with stock numbers. - they are NOT simply consecutive

Enter your scores for the RI lines data into a Text file (eg. MacExcel,
or another text file) as follows:-
A = like Columbia parent
B = like Landsberg parent
H = heterozygote (if possible to score)
- = unscorable or no score

Marker names should start with a * followed by a letter (small or capital)
ie. *w23 and should have a single space or tab between each entry. Names
MUST BE LESS than EIGHT characters. Mapmaker, the programme we use for mapping,
is not case sensitive and so reads "a" and "A" as the
same thing. Please check the current names used for Loci, so as not to generate
conflicts.

5. Enter data into Mapmaker.

If the data is sent to NASC for mapping then this section is for
interest only. Mapmaker for the Macintosh and UNIX are somewhat different to run (Lander et al 1987). If the database of 67 markers is being used with these programs we
strongly suggest that you READ THE INSTRUCTION
MANUALS and DO THE TUTORIALS before attempting to run
the program.

Use of the Kosambi mapping function (as opposed to Haldane)
appears to give the best fit for recombination data generated in
Arabidopsis (Koorneef and Stam, 1992). Kosambi mapping
function takes into account the effects of interference; which
means that after one recombination event has occurred it is less
likely that a second one will occur in adjacent regions, in the same
generation.

Before entering data into Mapmaker one needs to add two lines
of information above the markers scores.
The top line indicates what type of mapping population the data is
from, the options depend on the version of Mapmaker being used
(see below). The second line indicates the numbers of
individuals, the number of markers scored and defines the
genotype symbols.

If Mapmaker V1.0 is being used the data is considered as coming
from an F2 segregating population. This requires using a LOD
value of 6.0 (default is 3.0) for the group and three-point
commands, to take account of the multiple rounds of
recombination. The top line of the data file should read:-

data type f2 intercross

If Mapmaker V2.0 is being used on a UNIX one can run the data
as coming from an RI population ( and therefore use the default
LOD of 3.0). The default genotypes for this version are
A=Columbia and B=Landsberg. The top line of the file should
read:-

data type ri self

The second line indicates the number of individuals in the
population (ie. 100), the number of markers being scored (ie. 46)
and defines the genotype symbols used in the mapping data (ie.
CLXYHU or AB-). The respective second lines will look
something like:-

(V1.0) 100 46 0 0 CLXYHU (V2.0) 100 46 0 AB-

(PLEASE CHECK the manual of the version of Mapmaker being
used that these lines are correct for that version, they do vary).

At the end the program will produce a list of the markers with the
centimorgan distances and recombination fractions between them.
On the Macintosh this can be converted into a map; this is also
possible on the UNIX but the map produced is not so
informative. If Mapmaker V1.0 has been used the the
centimorgan distances will have to be recalculated, they are
approximately two-fold too big as they were calculated for an F2
population. The formula is:-